中村 郁郎

Although homologous recombination-promoted knock-in targeting to monitor the expression of a gene by fusing a reporter gene with its promoter is routine practice in mice, gene targeting to modify endogenous genes in flowering plants remains in its infancy. In the knock-in targeting, the junction sequence between a reporter gene and an endogenous target promoter can be designed properly, and transgenic plants carrying an identical and desired knock-in allele can be repeatedly obtained. By employing a reproducible gene-targeting procedure with positive-negative selection in rice, we were able to obtain fertile transgenic knock-in plants with the promoterless GUS reporter gene encoding β-glucuronidase fused with the endogenous promoter of MET1a, one of two rice MET1 genes encoding a maintenance DNA methyltransferase. All of the primary (T0) transgenic knock-in plants obtained were found to carry only one copy of GUS, with the anticipated structure in the heterozygous condition, and no ectopic events associated with gene targeting could be detected. We showed the reproducible, dosage-dependent and spatiotemporal expression of GUS in the selfed progenies of independently isolated knock-in targeted plants. The results in knock-in targeted plants contrast sharply with the results in transgenic plants with the MET1a promoter-fused GUS reporter gene integrated randomly in the genome: clear interindividual variation of GUS expression was observed among independently obtained plants bearing the randomly integrated transgenes. As our homologous recombination-mediated gene-targeting strategy with positive-negative selection is, in principle, applicable to modify any endogenous gene, knock-in targeting would facilitate basic and applied plant research. © 2009 Blackwell Publishing Ltd.

Polymerase chain reaction (PCR)-based genome walking techniques are commonly used to clone unknown genomic regions flanking known sequences. However, these methods are typically problematic when applied to highly complex DNA templates isolated from plants with large genomes. Here we describe a reliable and efficient genome walking method that is particularly effective for plants with large genomes. Our ligation-mediated PCR method, Straight Walk, has improved sensitivity and specificity due to optimization of sequences of adaptors and adaptor primers. Successful genome walking in lily, which has one of the largest genomes in plants, indicates that Straight Walk is applicable for most plant species. © 2009 Elsevier Inc. All rights reserved.

Rice PolA1 gene, encoding for the largest subunit of RNA polymerase I, spans ca. 15 kb containing 21 exons and presents as a single-copy-per-haploid genome. The genus Oryza comprises 22 wild species and 9 recognized genome types: AA, BB, CC, EE, FF, GG, BBCC, CCDD, and HHJJ. We analyzed sequences of the 19th intron (PI19) within PolA1 genes in 17 Oryza species. The AA species, containing two cultivated species, showed similar length of PI19 to that of CC species (287-296 bp). The longer PI19s were found in BB (502 bp) and FF (349 bp) species, although EE (217 bp) and GG (222 bp) species had shorter sequences. The size differences of the PI19s are particularly useful to discriminate between diploid (BB and CC) and allotetraploid (BBCC) species using simple PCR analysis. The evolutionary relationship among seven genomes was inferred based on the comparison of their PI19 sequences. © Springer-Verlag 2009.

The section Sitopsis in the genus Aegilops includes five species, Ae. speltoides, Ae. longissima, Ae. sharonensis, Ae. searsii, and Ae. bicornis, which share the SS genome. Although extensive molecular studies have indi- cated Ae. speltoides as a donor of BB or GG genome to polyploid wheat species, the precise relationships among SS, BB, and GG genomes remain unclear. PolA1 is a single-copy nuclear gene encoding the largest subunit of RNA polymerase I. Highly polymorphic PolA1 exon 20 sequences were analyzed for 11 Triticum- Aegilops, 13 Hordeum and three related species. Phylogenetic analyses of the PolA1 gene showed that Triticum-Aegilops and Hordeum species were distinctly separated into two clades. Two related species, Secale cereale and Dasypyrum villosum, were grouped into Triticum and Hordeum clades, respectively. Interestingly, seven accessions of the Sitopsis species were clustered into the Hordeum clade whereas two accessions belonged to the Triticum clade. In contrast, all accessions of Sitopsis species shared the same haplotype of plastid PSID sequences with Triticum-Aegilops species. This inconsistency in phylogeny between nuclear and cytoplasmic sequences suggested that the Sitopsis species probably originated through introgres- sive hybridization between ancestral species of Triticum-Aegilops and Hordeum.

While the Arabidopsis genome carries one copy of the methyltransferase 1 (MET1) gene for DNA methyltransferase, which is mainly responsible for maintaining CpG methylation, the rice genome bears two copies of the MET1 genes, OsMET1a and OsMET1b. The transcripts of OsMET1b accumulate more abundantly than those of OsMET1a in all of the tissues examined, and both genes actively transcribed at the callus, imbibed embryo, root, meristem, young panicle, anther, pistil, and endosperm, all of which contain actively dividing cells. The OsMET1a transcripts contain two 5′-untranslated exons and alternatively spliced 3′-terminal exons. The alternatively spliced transcripts consist of 14, 15, or 16 exons, and all of them encode a putative protein of 1527 amino acids. While the 3′-terminal exon of OsMET1b is unique, alternative splicing occurs in the 5′-terminal regions, which comprise either exons containing 5′-untranslated regions or an exon bearing the initiation codon. Depending upon alternative usage of 5′ exons by alternative splicing, the OsMET1b transcripts comprise 11, 12, 13, or 14 exons, and the former two and the latter two longer transcripts encode putative proteins of 1486 and 1529 amino acids, respectively. Moreover, the 5′ splicing patterns of OsMET1b can vary in different tissues. These findings are discussed with respect to the possible regulation of the OsMET1 genes. © 2007 Elsevier GmbH. All rights reserved.

The genus Kalanchoe is currently divided into section Kalanchoe and section Bryophyllum, and there has been no successful report on the production of inter-sectional hybrids. Therefore, reciprocal crosses were made between Kalanchoe spathulata (sect. Kalanchoe) and K. laxiflora (sect. Bryophyllum) in order to obtain basic information on the reproductive barriers between these two sections. The seeds were aseptically germinated in vitro and the plants were grown in greenhouse till flowering. When K. spathulata was used as a maternal donor, 39 out of 80 plants showed intermediate characteristics between K. spathulata and K. laxiflora. In contrast, no plants were obtained in the reverse crosses. Hybridity of these plants was confirmed by flow cytometric analysis, chromosome numbers and RAPD analysis. Bulbil formation on the leaf margin as one of the conspicuous characteristics of K. laxiflora was not observed in the hybrids. Some of the hybrid lines showed some pollen fertility, but failed to yield viable seeds by self-pollination or backcross-pollination. Successful production of the inter-sectional hybrid between the two species suggests that they are not so distantly related as considered previously. © 2007 Springer Science+Business Media B.V.

Chocolate cosmos has chocolate-coloured flowers with a scent of chocolate, and leaves like miniature dahlias. Chocolate cosmos is endangered in its native Mexico. It has been classified ambiguously as Cosmos atrosanguineus or Bidens atrosanguinea. We have resolved the phylogenetic relationship of chocolate cosmos among the genera Cosmos, Bidens and Dahlia by an analysis of plastid subtype identity (PSID) sequences. PSID sequences snowed that chocolate cosmos has a closer relationship with Cosmos than with Bidens or Dahlia. Based on this finding, chocolate cosmos was open-pollinated with two Cosmos species, C. bipinnatus and C. sulphureus. We successfully produced a hybrid plant with C. sulphureus using embryo rescue. The hybrid plant showed an intermediate phenotype such as crimson-red flowers. Its hybrid nature was confirmed by DNA fingerprinting and flow cytometric analysis.

Wild species and cultivars of Petunia were subjected to analysis for clarifying the historical progenitors of garden petunias (Petunia hybrida) using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of Chalcone synthase J (Chs-J) gene. The PCR products for Chs-J intron with adjacent part of exons digested with RsaI enzyme revealed that P. integrifolia and P. inflata (P. integrifolia complex), both with purple flowers, had one large-sized band [650 base pairs (bp)], whereas P. axillaris (P. axillaris complex), a white-flowered species, showed two smaller-sized bands (200 or 280 bp and 350 bp). In P. axillaris, two different band patterns were found among the three subspecies: 200 and 350 bp for P. axillaris subsp. axillaris and 280 and 350 bp for P. axillaris subsp.parodii and subsp. subandina. The 200-bp band was revealed to be specific to P. axillaris subsp. axillaris. P. hybrida cultivars showed four different band patterns, each of which consisted of two to three of the four bands (200, 280, 350, and 650 bp) detected in the wild taxa examined. These results indicate that the wild species analyzed here might partially have contributed to the Chs-J gene of garden petunias analyzed and demonstrate the use of PCR-RFLP in establishing relationships among closely related species and cultivars of Petunia. The puzzling problem related to the possible contribution of more than one subspecies of P. axillaris is discussed.

Petunia axillaris complex and P. integrifolia complex consist of three subspecies (ssp. axillaris, ssp. parodii and ssp. subandina) and two species (P. integrifolia and P. inflata), respectively. Since these taxa within each complex can only be distinguished by the flower morphology, it would be useful to discriminate them by DNA markers. In this study, we sequenced the 19th intron and the 20th exon of the PolA1 gene, a single gene encoding the largest subunit of RNA polymerase I, and showed clear differences between P. axillaris complex and P. integrifolia complex, and also among all the taxa within each complex. These sequence variations will be promising to develop PCR-based markers to discriminate taxa within P. axillaris complex and P. integrifolia complex.

Oryza diploid species comprise AA, BB, CC, EE, FF, and GG genome groups. Cultivated rice, O. sativa L. of the AA genome, has two subspecies, indica and japonica. These two subspecies are differentiated in many characteristics, such as the number of nucleolar sites, annual-perennial habitat and polymorphism of plastid DNA. Although many researchers accept that japonica must originate from O. rufipogon, the evolutionary route of indica remains unclear. A 69-bp deletion in ORF100 is useful to discriminate between indica and japonica cultivars and between O. nivara and O. rufipogon; furthermore, PSID sequences of O. sativa-O. rufipogon complex contain hyper-variable sites consisting of C and A mononucleotide repeats, which are useful to classify wild accessions and cultivars into the six subtypes of 6C7A and 7C6A (japonica and O. rufipogon), 6C8A and 7C7A (O. nivara), 8C8A and 9C7A (indica). The 69-bp deletion is shared with O. nivara (6C8A, 7C7A) and indica (8C8A, 9C7A) but the 8C8A subtype is found only in indica. To elucidate variations of plastid DNA in the genus Oryza and cytoplasmic origin of indica, we compared two plastid sequences, ORF100 (676 bp) and PSBZ (629 bp), in 40 accessions of 14 Oryza diploid species. Sequence comparison showed that FF and GG genome species were distantly related to AA genome species. Both BB and CC genome species were related with AA genome species but were mutually differentiated. An evolutionary trace of ORF100 and PSBZ sequences indicated that O. barthii might have a consensus sequence among all AA genome species. Both O. longistaminata and O. glumaepatula were highly related with O. barthii; the O. sativa-O. rufipogon complex showed more various polymorphisms than other AA genome species. The results of this study show that 8C8A and 9C7A subtypes of plastids in indica cultivars are differentiated from 6C8A and 7C7A subtypes in O. nivara, despite sharing the same deletion in ORF100. In addition, 6C7A and 7C6A subtypes in japonica cultivars and O. rufipogon share identical sequences in PSBZ but differentiate at two SNP sites in ORF100.

Kalanchoe blossfeldiana (sect. Kalanchoe) is one of the most important ornamental indoor plants during the winter season. Breeding of K. blossfeldiana has been restricted to intra and interspecific hybridization with allied species. To enlarge the genetic variability, reciprocal crosses were carried out between K. blossfeldiana and several wild Kalanchoe species which belong to sect. Kalanchoe and sect. Bryophyllum, and ovule culture technique was applied to rescue the hybrid embryos. Hybridity of the F1 plants thus obtained was confirmed by flow cytometric and RAPD analyses. Consequently, 6 intra-section and 3 inter-section cross combinations were obtained in the present study. Reciprocal compatibility was observed in the crosses with 2 wild species. All the other hybrids could be obtained when K. blossfeldiana was used as maternal donor. Flower morphology and color of these hybrids were almost intermediate between both parental species. Since these F1 hybrids were sterile, amphidiploids of these hybrids were produced to restore the fertility by utilizing spontaneous chromosome doubling in tissue culture.

The dried roots of Angelica acutiloba Kitagawa var. acutiloba Kitagawa, A. acutiloba Kitagawa var. iwatensis Hikino and A. acutiloba Kitagawa var. sugiyamae Hikino have been used as the herbal medicine known in Japan as Japanese Angelica Root. The respective morphological features of, in particular, A. acutiloba var. sugiyamae and A. acutiloba var. iwatensis are similar, and they are not easy to distinguish morphologically from each other. In an attempt to find a method for discriminating among these three varieties, we compared the nucleotide sequence of the spacer region between the atpF and atpA genes among the respective plastid genomes. Comparison of these sequences allowed us to identify each of the three varieties unequivocally. © Georg Thieme Verlag KG Stuttgart.

The purpose of this study was to assess the extent of genetic erosion of traditional upland germplasm in northern Thailand as a result of gene-flow from distinct strains carrying different genotypes. Even modern variety specific markers have not been developed, there is a comparative population in Laos. Thus, both populations were compared with various characters to evaluate gene-flow from modern variety to landraces. Glutinous and glabrous strains are predominated in Laos. However, such strains were drastically decreased in north-east Thailand. Gene diversity is higher in Thailand, compared to Laos at seven isozyme loci. This was a result of the higher frequencies of Indica strains and heterozygotes in Thailand. Plastid type was also determined by using an INDEL marker. Nearly half of Indica strains carried the Japonica plastid. Heterozygotes also tended to carry Japonica cytoplasm. Such nuclear-cytoplasm substituted strains and heterozygotes were probably generated by natural hybridization. Japonica strains tended to be a maternal donor rather than Indica ones. Or Indica strains would easily release pollens, which grow outside of upland fields. © Springer 2006.

Genetic variations among flowering cherries (Prunus subgenus Cerasus) were analyzed by spacer sequences between ribosomal protein L16 (rpl16) and ribosomal protein L14 (rpl14) genes of chloroplast DNA, these sequences were named plastid subtype ID (PS-ID), by using a total of 40 individuals from 11 species and 3 cultivars. Nucleotide sequences of ca. 420 bp were identified as part of rpl16 gene and PS-ID regions. One mutation site was found in partial nucleotide sequences of rpl16 gene. Five different A-repeat types were found at PS-ID region, which were denoted as 9A-T-10A, 10A-T-9A, 13A, 14A, and 15A, respectively. One base change also existed in the downstream of A-repeat. Many individuals (20/22) in species that originated from Japan, except for P. pendula f. ascendens, were 14A type, whereas all 9 individuals of P. pendula f. ascendens were 10A-T-9A type. Therefore, the maternal line of cultivars related to P. pendula f. ascendens can be revealed by the analysis of PS-ID region. In addition, P. pendula f. ascendens differs from other Japanese taxa based on morphological traits. The difference is supported from the nucleotide sequences of PS-ID in this study. The A-repeat types of cultivars, i.e., 'Someiyoshino', 'Ichiharatoranoo', and 'Shirotae', were 10A-T-9A type, 14A type, and 14A type, respectively, which suggests that the female parent of the 'Someiyoshino' was P. pendula f. ascendens. The results of 'Ichiharatoranoo' and 'Shirotae' analyses were not contradictory to the morphological taxonomy. PS-ID region was highly variable and useful for evaluating genetic variation and elucidating the origin of cultivars.

Dried aerial parts of Scutellaria galericulata L. (Labiatae; marsh skullcap) and Scutellaria lateriflora L. (Labiatae; mad dog skullcap) are mainly used as skullcap, a medicinal herb, in Europe and the United States. The respective dried aerial parts of the two species are difficult to distinguish morphologically from each other. We attempted to discriminate among six species (Scutellaria altissima L. (Labiatae), Scutellaria baicalensis Georgi (Labiatae), Scutellaria galericulata, Scutellaria incana Spreng. (Labiatae), Scutellaria indica L. (Labiatae) and Scutellaria lateriflora) of Scutellaria, which include three medicinal species (Scutellaria galericulata, Scutellaria lateriflora and Scutellaria baicalensis), by comparing the respective nucleotide sequences of the plastid rpl16 gene and the rpl16-rpl14 spacer region. Comparisons of these sequences allowed us to identify each of the six species unequivocally. © 2005 Elsevier Ireland Ltd. All rights reserved.

A quantitative trait locus (QTL) associated with the protein content of cytosolic glutamine synthetase (GS1; EC 6.3.1.2) in senescing leaves, panicle number, and panicle weight was characterized in rice (Oryza sativa L.). A near-isogenic line (NIL), C-22, developed by marker-assisted selection was grown under different nitrogen levels in the greenhouse and in a paddy field. Chromosome 2 of C-22 had an approximately 50-cM segment substituted from the Kasalath (indica) chromosome in a Koshihikari (japonica) genetic background. C-22 showed a 12-37% lower content of GS1 protein in leaf blades than Koshihikari, which was in good agreement with a QTL region positively affected by the japonica chromosome. At an early vegetative stage, C-22 had more active tillers than Koshihikari in the greenhouse. At the reproductive stage, both panicle number and total panicle weight of C-22 were significantly higher than those of Koshihikari, particularly when the plants were grown under a low-nitrogen condition. These traits of C-22 were further confirmed in a paddy field. Thus, tiller development was positively affected by the Kasalath chromosome at an early vegetative stage, which resulted in an increased panicle number and panicle weight at the mature stage in C-22. These data indicate that the target QTL (Pnn1; panicle number 1) is important in the development of tillers and panicles in rice. Linkage analyses for panicle number and ratio of developing tiller formation in the second axil (RDT) revealed that Pnn1 was delimited at the 6.7-cM region. © Springer-Verlag 2004.

Common non-waxy (Wx) rice cultivars contain two different alleles at the waxy locus, designated Wxa and Wxb, which encode different levels of granule-bound starch synthases and are hence involved in the control of endosperm amylose content. The Wxa allele was predominant in non-waxy indica cultivars, whereas the Wxb allele was common to the non-waxy japonica variety. Recently, some of the molecular mechanisms underlying the differentiation of Wxa from Wxb have been characterized. One structural difference between these two alleles was shown to be due to alternative splicing caused by a single-base substitution (AGGT to AGTT) at a donor site of the first intron within the Wx gene. In the case of waxy (wx) rice, it was not possible to distinguish whether the each wx allele was derived from Wxa or Wxb alleles by phenotypic analysis. However, we succeeded in developing a derived cleaved amplified polymorphic sequence (dCAPS) marker for the detection of the one-base splicing mutation without the need for sequencing. A mismatch primer was used to generate a restriction site in the Wxa allele (AGGT) but not in the Wx b allele (AGTT). Three hundred fifty-three waxy rice strains that are widely found in Asia were then employed for analysis using this dCAPS marker. Our findings suggested that waxy rice strains have both Wxa-and Wxb-derived alleles, but that the Wxb-derived allele was predominant, and its distribution was independent of indica-japonica differentiation. The wild relatives of cultivated rice all possessed the AGGT allele. It was concluded that the waxy mutations, and the corresponding rice cultivation, originated from japonica during the evolution and domestication process of rice and was preferentially selected by most Asian peoples. © Springer-Verlag 2004.

An attempt was made to discriminate among six species of Papaver (P. bracteatum, P. orientale, P. pseudo-orientale, P. rhoeas, P. setigerum and P. somniferum) by comparing the nucleotide sequences of the plastid rpl16 gene and the rpl16-rpl14 spacer region. Comparison of sequences allowed us to distinguish five species, namely P. bracteatum, P. orientale, P. pseudo-orientale, P. rhoeas and P. setigerum plus P. somniferum from one another, but sequences from P. setigerum and P. somniferum were identical. It is difficult to distinguish between P. bracteatum, P. orientale and P. pseudo-orientale at the vegetative stage of growth. However, our method allowed us to distinguish between these three species and the others using nucleotide sequences and should allow identification of P. bracteatum that has been cultivated illegally in the garden in Japan. Furthermore, P. rhoeas was clearly discriminated from P. setigerum and P. somniferum by reference to the sequence of the rpl16 exon using young seedlings. © 2003 Elsevier Ireland Ltd. All rights reserved.

The direct ancestor of rice (Oryza sativa L.) is believed to be AA genome wild relatives of rice in Asia. However, the AA genome wild relatives involve both annual and perennial forms. The distribution of the retrotransposon p-SINE1-r2, a short interspersed nuclear element (SINE) at the waxy locus was analyzed in diverse accessions of the AA genome wild relatives of rice (O. rufipogon sensu lato). Most annual wild rice accessions had this retrotransposon, while most perennial types lacked this element, contradicting results to the previous studies. Results presented here suggest that O. sativa has dual origin that lead to indica-japonica differentiation. Results suggest the indica line of rice varieties evolved from the annual genepool of AA genome and the japonica varieties from the perennial genepool of AA genome wild rice.

A single nucleotide polymorphic sequence (SNP) in the trnL-F intergenic spacer region of chloroplast DNA that discriminate Musa acuminata (AA) cytoplasm from Musa balbisiana (BB) cytoplasm has been found. This mutation was initially amplified using a pair of universal primers and converted into dCAPS (derived cleaved amplified polymorphic sequence) markers. Using these markers in combination with flow cytometric analysis, 'Pisang Klutuk' (syn. 'Pisang Awak') was found to be a triploid ABB cultivar (not a wild BB accession as previously classified). A 15-bp long deletion was also found within the trnL-F spacer sequence of M. acuminata subspecies banksii. This mutation will be useful as a specific marker for the cytoplasm derived from this subspecies. These maternally inheritable chloroplast DNA mutations discovered in this study are vital for better understanding the origins of banana cytoplasm to clarify the lineage of banana cultivars and the contribution of wild progenitors to cultivated types. © 2003, Japanese Society for Plant Cell and Molecular Biology. All rights reserved.

The banana (Musa sp., AAA) genome is continuously expanding due to the high frequency of somaclonal variation. Because of this increasing diversity, numerical and morphological methods of taxonomic and phylogenetic identification of banana cultivars became laborious, difficult, and often the subject of disagreements. The aim of this study, therefore, is to develop molecular tools for DNA fingerprinting that can discriminate Musa, AAA Cavendish subgroup cultivars. In this paper, we showed that the plastid-subtype identity (PS-ID) sequence of the noncoding region between rpl16 and rpl14 genes of plastid DNA was highly conserved except for single-base substitution and deletion. These differences separated the clones into three groups (G1, G2, and G3) and suggested that clones within groups are closely related maternally. Using arbitrary primer A13, we later identified negative RAPD markers A133.0 and A131.3 specifically for S4 (selection from Giant Cavendish subgroup, AAA) and S11 ('Morado' from 'Red' and 'Green Red' subgroup, AAA), respectively. Fragments corresponding to the missing bands were sequenced and used as templates to design new primers with overlapping sequences. Two of these primers, Ba3.0A and Ba1.3A, successfully generated positive markers consistently amplified as Ba3.0A0.8 and Ba1.3A0.6 for S4 and S11, respectively. It is proposed that the method just described can be a better alternative over screening more arbitrary primers in generating positive markers in cases when negative ones were already identified. Results of PS-ID subtype analysis likewise suggested potential use in identifying wild maternal progenitor in polyploid bananas.

We have identified two genes, located approximately 2.5 kb apart in the soybean genome, that are closely related to the α subunit of β-conglycinin. One of these genes has been shown to encode the α subunit and the other is very similar to the α subunit gene (referred to as the "α-related gene"). To determine whether the latter α-related gene expresses the α subunit, the structure of the chromosomal DNA region that contains the two genes was compared among soybean varieties exhibiting different expression levels of the subunit protein. We observed the presence of deletions of the α subunit and α-related genes in some varieties. In the naturally occurring variety Keburi, which accumulates the α subunit protein, the entire α-related gene was deleted in addition to the previously known deletion of the α′ subunit gene. On the contrary, the α subunit gene was deleted in Moshidou Gong 503 and Kari-kei 434. These structural analyses revealed that the α-related gene expresses the α subunit at least in Moshidou Gong 503 and Kari-kei 434. These two varieties do not harbour the α subunit gene, but carry the intact α-related genes and still express the α subunit.

To develop a rice cultivar that would be suitable for direct-seeding cultivation in cooler temperate regions, we generated transgenic rice plants in which a rice encoding a ζ-class glutathione S-transferase (GST) under the control of a maize ubiquitin promoter. GSTs have been suggested to be responsible for tolerance to various stresses such as cold, salt and drought by detoxification of xenobiotic compounds and reactive oxygen species. A total of 87 R0 transgenic rice plants harboring a chimeric GST gene were generated using Agrobacterium mediated transformation. Three R2 lines homozygous for the transgene were assayed for GST activity and had higher GST and glutathione peroxidase activities than non-transformants. Seedlings of the transgenic lines demonstrated greatly enhanced germination and growth rates at low temperature grown under submergence. The GST transgenic lines should be useful for breeding rice cultivars suitable for direct-seeding cultivation in cooler temperate regions.

Plastid subtype ID (PS-ID) sequences were determined from sequence data based on CA repeats between genes rpl16 and rpl14 in Japanese lowland and upland cultivars. The PS-ID sequences of Japanese rice cultivars showed that there are different maternal origins between lowland and upland cultivars. One subtype, 6C7A, of PS-ID sequences was predominant in all but one Japanese lowland cultivar and carried a combination of the indica-specific subtype 8C8A and japonica-specific nuclear markers for the isozyme genotype. It is probably a nuclear-cytoplasmic recombinant resulting from natural out-crossing and succeeding self-pollination. The origin of the plastid was re-confirmed by the existence of an indica-specific deletion in the plastid genome. In contrast, the Japanese upland cultivars showed two subtypes, 7C6A and 6C7A, of PS-ID sequences. An upland-specific isozyme allele as a nuclear marker was equally predominant in cultivars carrying each subtype. The existence of these particular upland-specific nuclear and cytoplasmic genotypes suggests that the origin of Japanese upland cultivars is different from that of Japanese lowland cultivars. Cultivars carrying the upland-specific nuclear genotype are common in Southeast Asia, but the combination of the upland-specific nuclear and cytoplasmic genotypes which is the same as the Japanese upland predominant type was found in cultivars only in Taiwan and Indonesia. Japanese upland cultivars are closely related to those cultivars.

Transgenic rice (Oryza sativa cv. Sasanishiki) overexpressing the wasabi defensin gene, a plant defensin effective against the rice blast fungus, was generated by Agrobacterium tumefaciens-mediated transformation. Twenty-two T2 homozygous lines harboring the wasabi defensin gene were challenged by the blast fungus. Transformants exhibited resistance to rice blast at various levels. The inheritance of the resistance over generations was investigated. T3 plants derived from two highly blast-resistant T2 lines (WT14-5 and WT43-5) were challenged with the blast fungus using the press-injured spots method. The average size of disease lesions of the transgenic line WT43-5 was reduced to about half of that of non-transgenic plants. The 5-kDa peptide, corresponding to the processed form of the wasabi defensin, was detected in the total protein fraction extracted from the T3 progeny. Transgenic rice plants overproducing wasabi defensin are expected to possess a durable and wide-spectrum resistance (i.e. field resistance) against various rice blast races.

Genetic variation of Japanese rice cultivars were examined. Five of 450 lowland cultivars and another five of 200 upland cultivars were determined as the indica type by using isozyme genotypes and the remainder were of the japonica type. The major characteristics of these indica cultivars, revealed a slender shape of grains, a short apiculus hair length, a positive allele for Ph reaction, and allele-3 for the Pgd1 locus. Three of these indica cultivars showed a non-deletion ORF100, which is essential to the japonica-type plastid. The plastid subtype identity (PS-ID) sequences of these plastids is 6C7A, which is also a japonica-specific repeat unit. Thus, these cultivars were concluded to be naturally generated cytoplasm substituted lines. These plastids were introduced into a indica genetic background from japonica cultivars grown elsewhere. The rest of the indica cultivars revealed a deletion-type ORF100 and plastid subtype 8C8A, both of which are indica-specific. These cultivars carried indica-type allelic constitutions for diagnostic isozyme loci. However, other characters were identical to the cytoplasm-substituted cultivars in Japan. In East and Southeast Asia, cultivars carrying a indica-type nuclear genotype with a japonica-type plastid are restricted to Aus cultivars in the Bengal region. Genetic and historical records suggest that Japanese indica cultivars and the Aus cultivars are closely related. The Aus cultivars acquire necessary genetic constitutions from both indica and japonica cultivars through naturally occurring out-crossing to adapt to a particular cultivation condition in the region. The wide adaptability enabled them to be introduced into a northern region like Japan.

A shoot overgrowth mutant of rice (Oryza sativa L.), accelerated internode overgrowth-1 (ao-1), is marked by accelerated longitudinal elongation of aerial parts and overgrowth of internodes at the vegetative stage. The physiological properties of ao-1 were similar to those of wild plants treated with a saturating level of exogenous gibberellins (GAs), except for the internode-overgrowth phenotype, which was not mimicked by GA-treated wild plants. The ao-1 mutant was less sensitive to a GA biosynthesis inhibitor, Uniconazole-P, than the wild type. Dwarf alleles of three loci, including two GA-sensitive and one GA-insensitive mutation, were introduced to produce double-mutants with ao-1, but the overgrowth phenotype was not suppressed in double-homozygous mutants. These results suggest that the overgrowth phenotype of ao-1 is caused by abolition of GA signaling rather than by GA overproduction. It is likely that a part of the shoot regulation system of ao-1 is saturated with the GA signal. As a possible model consistent with the results, we propose that AO-1 protein acts as a negative regulator in GA signal transduction. © The Botanical Society of Japan and Springer-Verlag 2002.

β-conglycinin, a soybean seed storage protein, is comprised of three different subunits, α, α', and β. Several candidates for the α subunit gene have been isolated, however, the structure of the α subunit gene has not been completely determined. Accordingly, it was also unknown which of the gene candidates are functionally active. Here, we have determined the nucleotide sequence and transcription start site of the α subunit gene, and compared the structural components with those of the other subunits or other seed protein genes. The α subunit gene, which is located on a 7.6-kb EcoRI fragment, was composed of six exons that had the same organization as those for the α' subunit gene. Within a 400 bp upstream region of the transcription start site, four regions (designated as boxes I, II, III, and IV) were found to be conserved among the α, α, and other seed protein genes. Genomic Southern blot analysis of soybean varieties lacking the α subunit gene candidate indicated that the gene characterized in this paper actually encodes the α subunit and is functionally active. In addition, these experiments revealed the presence of an additional gene which is also responsible for the expression of the α subunit.

An analysis of random amplified polymorphic DNA (RAPD) was performed using nine accessions of three species of medicinal plants in the genus Scutellaria (S. galericulata, S. lateriflora and S. baicalensis; known collectively as skullcap) in an effort to distinguish between members of these three species. Dried aerial parts of the two species S. galericulata and S. lateriflora are difficult to distinguish morphologically. Ten arbitrary primers produced 92 fragments, and eight of the primers yielded 23 species-specific fragments among the three species. Six fragments were specific for S. galericulata, seven for S. lateriflora and ten for S. baicalensis. When primers A02 and A06 were used in the polymerase chain reaction, RAPD fragments that were specific for each of the three species were generated simultaneously. Primer A02 produced five species-specific fragments: one was specific for S. galericulata; two for S. lateriflora; and two for S. baicalensis. Primer A06 produced three species-specific fragments: one for S. galericulata; one for S. lateriflora; and one for S. baicalensis. The RAPD markers that were generated with these two primers should rapidly identify members of the three species of Scutellaria. The consistency of the identifications made with these species-specific RAPD markers was demonstrated by the observation that each respective marker was generated from three accessions of each species, all with different origins. Furthermore, cluster analysis using the 92 RAPD fragments produced a dendrogram of genetic relatedness that was in good agreement with the taxonomic designations of the three species. Thus, the RAPD markers should be useful for the future identification of members of the three species of medicinal Scutellaria plants.

Three random amplified polymorphic DNAs (RAPDs) closely linked to the vrs1 (formerly v) locus were sequenced and converted to sequence-tagged sites (STSs). Of the three STSs, two retained the RAPD polymorphism as dominant-recessive markers between 'Kanto Nakate Gold' (KNG; a two-rowed barley) and 'Azumamugi' (AZ; a six-rowed barley), while the other was co-dominant after digestion with restriction enzymes. Six restriction fragment length polymorphisms (RFLPs) linked to vrs1 were converted to six STSs. All six STSs were co-dominant between the two cultivars after digestion with restriction enzymes. A reliable protocol for small-scale DNA isolation from leaf tissue was developed. The STS markers and the small-scale DNA isolation protocol developed in this study are useful tools for mapping the vrs1 locus of barley.

Recent complete sequence data of the cyanobacteria genome and the plastid genome of algae and land plants clearly show that plastids originated from the endosymbiotic integration of a photosynthetic prokaryote into eukaryotic host cells. One indication of this event is that the organization of ribosomal protein genes has been conserved from bacteria to plastids of land plants. We are interested in the rpl16 and rpl14 genes, which are interrupted by the rpl29 and rps17 genes in both the cyanobacteria genome and the plastid genome of red alga, but are adjacent in liverwort to higher plants. This suggests that a linker between rpl16 and rpl14 was made by deletion of the rpl29 and rps17 genes at the appearance of land plants, and therefore the rpl16-rpl14 linkers of land plants have diverged from a common ancestral sequence. The rpl16 and rpl14 genes are highly conserved (more than 84 % homology) among rice, spinach and tobacco, whereas their linkers show low homology (28 %). The plastid rpl16-rp114 linker sequences of higher plants, such as barnyard grass, gentian and oak tree could be amplified and sequenced using a pair of common primers, even if their sequences are unknown. We propose short DNA sequence in the 3' noncoding region from the stop codon of the rpl16 gene as plastid subtype ID sequence (PS-ID). PS-IDs of ca. 50 bp are thought to be long enough to address plastid subtypes of higher plants and the establishment of a PS-ID database could contribute to plant phylogeny.

Recombinant backcross lines of barley were produced from a cross between Kanto Nakate Gold (KNG; two-rowed) and Azumamugi (AZ; six-rowed) after backcrosses of F1 plants with AZ as the recurrent parent. Each of these lines had an introgressed segment from chromosome 2 of KNG. Two recombinant backcross lines, L1 and M3-13, were used for an initial screening of polymorphism. After screening a total of 888 oligonucleotides as arbitrary primers, we identified eight random amplified polymorphic DNAs (RAPDs) between backcross lines and AZ. Among the RAPD fragments, CMNA-38700 was linked to the v locus with a recombination frequency of zero, while OPJ-09850 and OPP-02700 were linked to the v locus at a map distance of 1.4 cM. Thus, the three RAPD markers were clustered around the v locus since the lengths of introgressed chromosomal segments in the L1 and M3-13 lines were no less than 38 cM. The other five RAPD fragments that we identified were not linked to the v locus.

From a cDNA library of cultured Glycyrrhiza echinata L. (Fabaceae) cells treated with yeast extract (YE) elicitor, eight cytochrome P450 (P450) fragments (Ge-1 to 8) were isolated using the PCR method based on conserved sequences of P450s. Comparison of amino acid sequences revealed that two of the fragments (Ge-1 and 2) belong to the CYP73 family encoding trans-cinnamic acid 4-hydroxylase. Ge-4 had 63% identity with the sequence included in CYP93, which has recently been reported to be induced by methyl jasmonate in soybean cells (Suzuki et al., FEBS Lett., 383 (1996) 836 86), and Ge-6 and 7 were highly homologous to the partial sequence of CYP84 encoding ferulic acid 5-hydroxylase of Arabidopsis thaliana (Meyer et al., Proc. Natl. Acad. Sci. USA, 93 (1996) 6869-6874). Others (Ge-3, 5 and 8) displayed homology less than 43% with known plant P450s. A full-length cDNA (CYP73A14) containing Ge- 1 sequence was isolated from the cDNA library, and its amino acid sequence showed 91 93% identity with CYP73 of other leguminous plant species. Northern blot analysis of mRNAs of G. echinata cells indicated that CYP73 (Ge-1 and 2) genes were transcribed at the early stages post-elicitation (< 10 h). The transcription level of the Ge-1 gene was much higher than that of Ge-2. Transcription of Ge-3, 5, 6, and 7 was also activated by elicitation, but no clear signal from Ge-4 and 8 was observed. When total RNAs of cultured cells of Medicago sativa were analyzed by Northern blotting, transcripts hybridized with G. echinata cDNA clones consisting Ge-1, 3, 5, and 6 sequences could be detected. Possible involvement of cloned P450s in elicitor-induced phenylpropanoid/flavonoid biosynthesis is discussed.

Transgenic rice plants harboring the bialaphos-resistant (bar) gene expressed from the maize ubiquitin promoter were inoculated with mycelia of the rice blast disease pathogen Magnaporthe grisea. Lesions caused by this fungal pathogen were decreased on the leaves of the transgenic rice plants following treatment with bialaphos. Pre-treatment of plants with bialaphos 1 day before the inoculation also suppressed disease symptoms to 20% of that in the control. Therefore, it may therefore be possible to control rice blast pathogen by the use of bar-transgenic rice plants along with bialaphos treatment.

In a previous study, we compared chloroplast DNAs (ctDNAs) from four species of Oryza and detected two independent deletions of DNA fragments in the ctDNAs (Kanno and Hirai 1992a). These deletions were genotype-specific variations. Since short direct-repeat sequences were detected at the borders of both deletions, the deletions were apparently the result of intramolecular recombination mediated by these direct-repeat sequences. In the present study, we examined whether or not this type of variation exists within a single species. Ishii et al. demonstrated three types of ctDNA in O. Sativa (1988), and the source of the variations that they identified seemed to be deletions. We determined the precise locations of the deletions and the sequences around them. As expected, our results showed that these variations were the results of deletions that were mediated by short direct-repeat sequences. While the deletions that had been found previously were located on spacer regions, those found in this study were located within open reading frames (ORFs). Northern hybridization analysis showed that one of the ORFs was-transcribed. In the case of this deletion, the amino acid sequence encoded by the C-terminal region of the ORF was altered and the short inverted-repeat sequences downstream of the ORF were deleted. In addition, there were other short inverted-repeat sequences downstream of the altered ORF. © 1993 Springer-Verlag.

Ninety Chinese rice landraces were examined with special reference to the indica-japonica differentiation in terms of traditional criteria, isozyme analysis and PCR analysis of the chloroplast DNA (cpDNA). Cultivars were separated into indica and japonica defined by a discriminant function (Z) based on key characters, as well as by isozyme genotypes. Most indica landraces had chloroplast DNAs with a deletion at the Pst-12 fragment, while most japonica landraces had cpDNAs without the deletion. Two traditionally recognized varietal groups in China, keng and hsien, corresponded largely to the respective japonica and indica revealed in our study. The results obtained in this study showed good agreement for classification of indica and japonica types by the three methods: discriminant analysis by Z value, isozyme analysis, and PCR analysis for cpDNA. © 1994 Kluwer Academic Publishers.

The RNA-dependent RNA polymerase associated with rice stripe virus was dissociated from viral RNA (vRNA) by CsCl centrifugation. The solubilized RNA-free RNA polymerase transcribed a model RNA template 50 nucleotides in length carrying the 5'- and 3'-terminal conserved sequences of all four genome RNA segments. A 3'-terminal half molecule of the model template was also active as a template. Hence, we propose that the 3'-terminal conserved sequence serves as a promoter for the rice stripe virus-associated RNA polymerase. The solubilized enzyme, however, was unable to transcribe vRNA. The failure of the solubilized enzyme to transcribe vRNA is discussed in relation to the apparent loss of RNA polymerase activity after treatment of virions with high concentrations of salt.